Estradiol mediates greater germinal center responses to influenza vaccination in female than male mice

ABSTRACT Adult females of reproductive age develop greater antibody responses to inactivated influenza vaccines (IIV) than males. How sex, age, and sex steroid concentrations impact B cells and durability of IIV-induced immunity and protection over 4 months post-vaccination (mpv) was analyzed. Vaccinated adult females had greater germinal center B cell and plasmablast frequencies in lymphoid tissues, higher neutralizing antibody responses 1–4 mpv, and better protection against live H1N1 challenge than adult males. Aged mice, regardless of sex, had reduced B cell frequencies, less durable antibody responses, and inferior protection after challenge than adult mice, which correlated with diminished estradiol among aged females. To confirm that greater IIV-induced immunity was caused by sex hormones, four core genotype (FCG) mice were used, in which the testes-determining gene, Sry, was deleted from chromosome Y (ChrY) and transferred to Chr3 to separate gonadal sex (i.e., ovaries or testes) from sex chromosome complement (i.e., XX or XY complement). Vaccinated, gonadal female FCG mice (XXF and XYF) had greater numbers of B cells, higher antiviral antibody titers, and reduced pulmonary virus titers following live H1N1 challenge than gonadal FCG males (XYM and XXM). To establish that lower estradiol concentrations cause diminished immunity, adult and aged females received either a placebo or estradiol replacement therapy prior to IIV. Estradiol replacement significantly increased IIV-induced antibody responses and reduced morbidity after the H1N1 challenge among aged females. These data highlight that estradiol is a targetable mechanism mediating greater humoral immunity following vaccination among adult females. IMPORTANCE Females of reproductive ages develop greater antibody responses to influenza vaccines than males. We hypothesized that female-biased immunity and protection against influenza were mediated by estradiol signaling in B cells. Using diverse mouse models ranging from advanced-age mice to transgenic mice that separate sex steroids from sex chromosome complement, those mice with greater concentrations of estradiol consistently had greater numbers of antibody-producing B cells in lymphoid tissue, higher antiviral antibody titers, and greater protection against live influenza virus challenge. Treatment of aged female mice with estradiol enhanced vaccine-induced immunity and protection against disease, suggesting that estradiol signaling in B cells is critical for improved vaccine outcomes in females.

vaccination and protection from live influenza virus infection are reduced (3,7,8), with evidence that the age-associated decline in immunity is greater for females than males in response to seasonal influenza vaccines in humans (9), the pandemic monovalent 2009 H1N1 vaccine in humans (3), and universal influenza vaccine candi dates in mice (10).Several studies illustrate that the effectiveness of the influenza vaccine decreases over an influenza season, likely due to waning levels of virus-specific antibodies (11)(12)(13), but whether age and sex influence the waning of influenza vaccineinduced antibody responses and protection has not been reported.
Greater vaccine-induced immunity and protection among adult females appear to be mediated by differential regulation of genes associated with B cell function.Tolllike receptor 7 (Tlr7) plays an important function in antibody isotype switching and antibody production in the germinal centers (GC) (14,15).Adult female mice have greater expression of the X-linked Tlr7 gene in splenic B cells following vaccination as compared to adult males, with deletion of Tlr7 eliminating sex differences in vaccineinduced immunity and protection (4).Increased DNA methylation in the promoter of Tlr7 contributes to greater Tlr7 expression in B cells from vaccinated female than male mice (4), but with known and putative estrogen response elements in the promoter of Tlr7 (16), regulation of Tlr7 expression by estrogen receptor signaling cannot be ruled out.
Expression of activation-induced cytidine deaminase (Aicda) mRNA, the gene that encodes activation-induced deaminase (AID) enzyme, is involved in somatic hypermuta tion (SHM) and shows greater expression in splenic B cells isolated from vaccinated adult females than adult male mice, with deletion of Aicda eliminating sex differences in vaccine-induced immunity and protection (6).These data suggest that sex differences in humoral immunity are dependent on greater class switch recombination (CSR) and SHM in B cells from female than male mice.Regulation of these processes in B cells by sex steroids has been established in autoimmune disease mouse models (17,18) but less so in the context of inactivated vaccines, where humoral immunity is the correlate of protection (19).Both in humans and mice, estradiol is positively, and testosterone is negatively associated with antibody titers after influenza vaccination (2,3).Moreover, in adult mice, sex differences in vaccine-induced immunity are eliminated by the removal of the gonads and restored by exogenous sex steroid replacement in gonadectomized male and female mice (3).The contributions of gonadal sex versus sex chromosome complement to sex differences in influenza vaccine-induced immunity and protection have not been systematically investigated.Because the estrogenic changes with aging affect vaccine-induced immunity (3), we hypothesized that sex steroids more than sex chromosome complement would mediate sex differences in influenza vaccine-induced immunity and protection against infection.Whether changes in sex steroid concentra tions affect the numbers of antibody-producing B cells, titers of antiviral antibodies, or both was further explored.Finally, consideration was given to the therapeutic use of estrogen replacement therapy for improving vaccine-induced immunity and protection in aged female mice.

Vaccinated adult females have greater numbers of antibody-producing B cells, antibody responses, and protection against influenza than males, which change with advanced age
Previous studies from our group reveal that after receipt of an inactivated 2009 H1N1 vaccine, adult females have greater neutralizing antibody responses, more cross-reactive IgG antibodies, more GC B cells in spleens, and greater SHM frequencies in regions of the recombined V genes in splenic GC B cells than males (3,4,6).Whether these sex differences in B cells change with aging has not been explored.Adult (2-3 month old) and aged (17 months old) male and female C57BL/CR mice were vaccinated twice with inactivated 2009 H1N1 vaccine in a 3-week interval.At 35 days post-vaccination (dpv), i.e., 14 days post-boost, draining lymph nodes (i.e., inguinal and popliteal) (20) were collected from vaccinated animals, and frequencies and total numbers of CD4-B220 + CD38 GL7 + GC B cells and CD4-B220 + CD138 + plasmablasts were determined by flow cytometry (Fig. 1A).Adult mice had significantly greater numbers of total lymph node cells and total B cells in lymph nodes than aged mice, and among adult but not aged mice, females had significantly greater numbers of these cells compared with males (Fig. 1B and C).Likewise, adult mice had significantly greater frequencies and numbers of GC B cells (Fig. 1D and E) and plasmablasts (Fig. 1F and G) in their draining lymph nodes than aged mice.Adult females had significantly greater numbers of GC B cells (Fig. 1E) as well as greater frequencies and numbers of plasmablasts (Fig. 1F and G) in draining lymph nodes than adult males.Sex differences in B cell numbers and proportions were not observed in lymph nodes from aged mice (Fig. 1D through G).The frequencies and numbers of GC B cells were also determined at 35 dpv in the spleen.As observed in the lymph nodes, the frequencies and numbers of GC B cells in the spleens of vaccinated mice were greater among adult than aged mice, with adult females having more GC B cells than adult males (Fig. 1H and I).Splenic GC B cells were sorted, and the J H 4 intronic regions of the recombined V genes were sequenced.Consistent with previous results (6), the mutation frequency in the J H 4 intronic region showed a trend of greater frequencies in splenic GC B cells from adult females than adult males (Fig. 1J, P = 0.1 with two-way ANOVA, P < 0.05 in T test), with the sex difference in SHM not observed among aged animals who generally had greater variability in SHM frequencies in splenic GC B cells than among adult animals (Fig. 1J).These data illustrate that adult females have greater numbers of GC B cells and plasmablasts in lymphoid tissues and greater SHM frequencies in GC B cells than adult males, which is mitigated with aging.
In addition to having more antibody-producing cells in draining lymph nodes and spleens at 1 mpv, vaccinated adult females had greater titers of anti-2009 H1N1-specific IgG, IgG2c, and virus-neutralizing, but not IgG1, antibodies than adult males (Fig. 2A through D).While adult mice had greater antibody responses than aged mice, no sex differences in antibody titers were observed among aged mice (Fig. 2A through D).Concentrations of estradiol were greater in adult females than in either males or aged females (Fig. 2E), reflecting the patterns observed for both antibody-producing cells and antibody titers.In contrast, adult males had greater testosterone concentrations than either females or aged males (Fig. 2F), which did not reflect the patterns of vaccine-induced immunity.These data suggest that the numbers of antibody-producing B cells and titers of antiviral antibodies are greatest in the animals that have the highest circulating concentrations of estradiol.

Sex differences in vaccine-induced antiviral antibody responses are durable over time among adult, but not aged, mice
To explore sex and age differences in the durability of vaccine-induced antibody responses and protection, adult (2-3 months old) and aged (17 months old) male and female C57BL/6CR mice were vaccinated twice with inactivated 2009 H1N1 vaccine at a 3-week interval and followed for 4 mpv, and plasma samples were collected at each month to measure anti-2009 H1N1 antibody responses.Adult mice maintained highly detectable anti-2009 H1N1 IgG, IgG2c, and nAb titers for up to 4 mpv, with females maintaining greater antibody responses than males for the duration of the study (Fig. 3A  through C).In contrast, after 1 mpv, anti-2009 H1N1 IgG, IgG2c, and nAb titers fell below the limits of assay detection among aged mice, with no sex differences observed (Fig. 3A through C).Vaccinated adult and aged male and female mice were challenged with a 2009 H1N1 drift variant virus at either 1 or 4 mpv.Infectious virus titers were measured in the lungs at 3 days post-challenge (dpc) and were significantly lower among adult than aged mice, with adult females having lower pulmonary virus titers than either adult males or aged males and females both at 1 and 4 mpv (Fig. 4A and C).Subsets of mice were followed for 14 dpc for morbidity.At 1 mpv, vaccinated aged mice lost significantly more body mass compared with adult mice, with adult males losing more body mass than adult females after challenge with 2009 H1N1 drift variant virus (Fig. 4B).In contrast after live virus challenge at 4 mpv, sex differences in protection from the disease were not observed either in adult or aged animals, but adult mice were still better protected from morbidity than aged mice (Fig. 4D).Taken together, these data suggest that the greater vaccine-induced immunity and protection against infection, but not disease, in adult female mice were durable over time but lost with age.

Sex steroids more than chromosomal complement cause sex differences in influenza vaccine-induced antibody responses and protection
Our previous work illustrated that adult females develop greater 2009 H1N1 vaccineinduced immunity and protection against 2009 H1N1 drift variant virus, which is mediated by both greater expression of the X-linked gene Tlr7 in B cells and estrogenic enhancement of immune responses (3,4,6).Our current work (Fig. 1 to 4) also indica ted that greater estradiol concentrations were associated with more durable antibody responses and protection against infection.To determine the contribution of sex steroids versus sex chromosome complement to sex differences in vaccine-induced immunity and protection, we used the FCG mouse model.The FCG mouse model involves deletion of Sry from chromosome Y (ChrY) and insertion of a Sry transgene on Chr3, resulting in XX gonadal females (XXF), XY gonadal females (XYF), XXSry gonadal males (XXM), and XY-Sry gonadal males (XYM).The immunity phenotype of these FCG mice can be compared in 2 × 2 experimental design to separate the contribution of gonadal sex and sex steroid (i.e., testes or ovaries that produce high concentrations of androgens or estrogens, respectively) from sex chromosome complement (i.e., XX or XY) (21).
Eight-to 10-week-old FCG C57BL/6 J mice were vaccinated twice with the inactiva ted 2009 H1N1 vaccine at a 3-week interval.Among gonadally intact adult FCG mice, estradiol concentrations were greater in gonadal females (XXF and XYF) than gonadal males (Fig. 5A), and testosterone concentrations were greater in gonadal males (XYM and XXM) than in gonadal females (Fig. 5B).At 28 dpv, we measured IgG and IgG2c binding to 2009 H1N1 as well as neutralizing antibody responses against the vaccine virus and observed that gonadal females (XXF and XYF) produce significantly greater antibody titers than gonadal males (XYM and XXM) (Fig. 5C through E).At 35 dpv, gonadal females (XXF and XYF) also had greater numbers of GC B cells and plasmablasts in the draining lymph nodes than gonadal males (XYM and XXM) (Fig. 5F and G).
Vaccinated FCG mice were challenged with a drift variant of 2009 H1N1 virus at 42 dpv and, 5 days later, euthanized to extract lungs to measure pulmonary titers of the virus.Gonadal females (XXF and XYF) had lower pulmonary titers of the virus than gonadal males (XYM and XXM) (Fig. 5H).A separate cohort of vaccinated and infected FCG mice was followed for morbidity (i.e., mass loss after infection) as a measure of how well vaccination protected not only against infection but also disease.Vaccine-induced protection against disease revealed gonadal sex by sex chromosome complement interaction, in which while gonadal males experienced greater disease than gonadal females, among gonadal females, XXF mice suffered significantly less morbidity than XYF mice (Fig. 5I).Taken together, these data suggest that sex steroids have a greater effect on vaccine-induced antibody-producing B cells and protection against infection than sex chromosome complement.

Estradiol supplementation in aged females improves influenza vaccineinduced antibody response and protection
Both the aging and FCG models illustrated that sex steroids are critical regulators of vaccine-induced humoral immunity and protection against infection with the influenza virus.If reduced estradiol concentrations, in particular, cause worse vaccine-induced immunity and protection, then estradiol supplementation in aged females might rescue immunity by improving antibody responses after vaccination and protection against infection.To test this hypothesis, adult (3-2 months old) and aged (17 months old) female C57BL/6CR mice were implanted either with placebo or estradiol-filled capsules and vaccinated with inactivated 2009 H1N1 vaccine and boosted after 3 weeks.At 35 dpv, IgG and IgG2c binding to 2009 H1N1 as well as neutralizing antibody responses against the vaccine virus were measured.Treatment of either adult (270.2 ± 13.66 pg/mL) or aged female mice (279.90 ± 28.78 pg/mL) with exogenous estradiol significantly increased concentrations beyond that of either adult (26.99 ± 5.9 pg/mL) or aged (12.02 ± 1.90 pg/mL) females treated with placebo (P < 0.05).The circulating concentrations of estradiol following pellet implantation were consistent with previous reports (22,23).In aged females, estradiol supplementation significantly improved anti-2009 H1N1 IgG, IgG2c, and neutralizing antibody responses after vaccination (Fig. 6A through  C).Specifically, vaccinated aged females with estradiol produced antiviral antibody responses that were comparable to adult females with either endogenous (i.e., placebo) or exogenous estradiol and were greater than aged females that received placebo treatment.Vaccinated adult and aged female mice were challenged with the 2009 H1N1 drift variant virus at 42 dpv, and infectious virus titers were measured in the lungs at 3 dpc.Estradiol supplementation in aged female mice did not significantly reduce replicating virus titers in the lungs of vaccinated mice as compared with aged females that received placebo treatment (Fig. 6D).In contrast, among mice that were followed for 14 dpc for morbidity, estradiol supplementation significantly reduced infection-induced morbidity as compared with placebo treatment in aged female mice (Fig. 6E).Vaccinated aged females treated with estradiol were as protected against severe influenza disease as vaccinated adult females that had either endogenous or exogenous estradiol.Taken together, these data highlight that estradiol replacement improves vaccine-induced antibody responses and reduces the burden of disease, but not virus replication, after infection in aged female mice.

DISCUSSION
Using diverse mouse models and hormone replacement, we explored how sex and aging impact the cellular mechanisms and durability of immunity and protection to inactivated influenza vaccine (IIV).Following vaccination, greater numbers and frequencies of GC B cells and plasmablasts, as well as antiviral antibody responses were associated with better protection against infection and disease following influenza virus challenge.The novelty of our work is that we show that elevated estradiol concentrations more than other biological factors are a strong predictor of better B cell-mediated immunity and protection against infection in females as compared with males.Loss of estradiol either through aging or in transgenic mice significantly impairs B cell immunity and long-term protection against influenza infection and disease.Both aged males and females had lower numbers of plasmablasts and GC B cells, less durable antibody responses, and reduced protection against both infection and disease following live virus challenge as compared with adult mice.Due to the waning of antibodies, influenza virus vaccine effectiveness declines significantly even within the same influenza season (11,24).Such antibody waning after influenza virus infection or vaccination is more prominent among older than younger adults (13,25).The age-associated decline in antibody responses can be broadly attributed to geriatric immunosenescence, with several B cell-specific defects associated (7,26).After vaccination, activated B cells undergo rapid proliferation and differentiation in the GCs within the secondary lymphoid tissues, including the spleen and lymph nodes (27).SHMs and class switch recombination occur within the GC and together underlie the production of high-affinity class-switched antibodies (28).Reduced serum antibody titers are observed among older compared to younger individuals after receipt of seasonal influenza vaccination, which is associated with lower numbers of plasmablasts (29).In humans receiving seasonal influenza vaccination, aged individuals have reduced SHM of plasmablasts as compared with younger aged individuals which results in an inability to mount antibody responses to the drifted epitopes of influenza virus (30).Reduced SHM was not observed with aging in our mice, which might reflect species-specific differences or kinetic differences in the timing of sample collection.
Sex chromosome complement (i.e., having XX or XY) can directly cause sex differences in a phenotype (e.g., humoral immunity) through an imbalance in the expression of X and Y genes that can affect immunity (31).For example, Tlr7 is encoded on the X chromosome and can escape X inactivation in immune cells from females (32).Though we did not measure levels of Tlr7 gene expression in this study, we previously showed that Tlr7 has greater expression in B cells from wild-type females than males following influenza vaccination (4).Sex chromosome complement also can indirectly cause sex differences in a phenotype by altering concentrations of sex steroids that can bind to nuclear receptors in immune cells to transcriptionally regulate immune cell function (33).For example, elevated testosterone in males dampens inflammatory (34) and antibody responses (2) to alter influenza virus infection and vaccination.There can also be combined effects of genes and hormones; some X-linked genes, e.g., Tlr7, contain estrogen response elements, and their expression can be regulated by sex steroids (16).Likewise, interferon regulatory factor (IRF) 5 is another relevant target of estradiol-medi ated regulation of B cells that needs further exploration.IRF5 has an important role in class switch recombination and GC formation, and its expression in B cells is positively regulated by estrogen signaling through estrogen receptor α (ERα) (35)(36)(37).
Using the FCG mouse model, we explored whether sex differences in humoral immunity to IIV are caused by direct effects of sex chromosome complement, effects of sex steroids on immune cell function, or both.Gonadal females, regardless of sex chromosome complement, had greater numbers of antibody-producing B cells and titers of vaccine-induced antibody, which led to better pulmonary virus clearance.In contrast, the combination of gonadal sex and sex chromosome complement appeared to modulate protection from severe disease, at least in females.Recently, it has been observed that a 3.2-MB region of the X chromosome is translocated to the Y chromo some with Sry deletion in the FCG mouse model, which results in the overexpression of various genes including Tlr7 in XY as compared with XX tissues (38).Interpretation of sex chromosome complement-mediated outcomes, in determining whether the difference in phenotype is caused by sex chromosome complement or by the higher expression of these translocated genes, is complicated by this new discovery.Our data highlight that gonadal sex more than sex chromosome complement causes sex differences in vaccine-induced immunity and protection.Future studies could use castrated FCG mice and B-cell-specific ERα knockout mice for further verification of the role that estrogen receptor signaling, specifically, plays in improving vaccine outcomes.
We showed that estradiol treatment can improve IIV-induced antibody production in aged females.Previous studies illustrate that estradiol treatment increases antibody response and protects adult female mice from influenza virus infection, and these effects are mediated through ERα signaling (23).In gonadectomized young adult mice, estradiol treatment restores antibody production after vaccination with an inactivated influenza virus split vaccine (39).Because B cells have estrogen receptors, estrogens, including estradiol, can transcriptionally regulate cellular activity and function (40), in part by binding to estrogen response elements in the promoter region of estrogen-responsive genes, such as Aicda, and directly activating AID transcription resulting in increased CSR and SHM (17).In contrast, testosterone suppresses splenic B cell function by downregu lating B cell activating factor, which is a cytokine essential for the survival of splenic B cells (41).Greater serum testosterone concentrations also are associated with reduced antibody response during malaria vaccination (42).
Estradiol treatment in aged females improved disease outcomes, but not virus replication, after influenza virus infection, indicating that estradiol treatment can rescue some, but not all, aspects of age-associated reductions in IIV-induced immunity.The inability of estradiol treatment to improve pulmonary virus clearance in aged mice is likely associated with age-specific changes in pulmonary integrity and function, which are irreversible with hormone treatment.For example, in aged female mice, influenza virus infection-induced inflammation promotes fibrosis to a greater extent than in adult female mice (43).Aged female mice also have neutrophils in the lungs with altered chemotactic gene expression and tissue localization, and lymphocytes with impaired effector and memory functions as compared with adult females (43).
Future studies should explore the role of sex steroids in the genetic and epigenetic regulation of GC B cell and plasmablast activity.Although AID enzyme activity was not measured in this study, the observation that IIV-specific IgG2c, but not IgG1, was greater among adult females than males and regulated by gonadal steroids highlights a fundamental role of biological sex differences in CSR.Future studies will need to consider the differential effects of gonadal steroids on the kinetics of the secretory functions of GC B cells and plasmablasts and B cell proliferation.
In humans, prior immunity caused by previous exposure to influenza viruses through infection or vaccination plays an important role in determining immune responses after subsequent influenza vaccination (44,45).In the current study, influenza-naïve mice were used, which does not incorporate the impact of pre-existing immunity on sex or age differences in vaccine effectiveness.High-dose or adjuvanted vaccines are recom mended in aged individuals for influenza, and we and others have shown that females maintain greater season-to-season antibody durability than males among individuals 75+ years of age (9).Whether high-dose or adjuvanted vaccines could overcome the deficiency in GC B cell and plasmablast numbers and functions in individuals with lower circulating estrogens should be explored.
Overall, our study highlights that estradiol is a biological factor contributing to improved outcomes to IIV vaccine.Future studies must consider how to harness this for adjuvants or other treatments to improve vaccine outcomes in post-menopausal Future studies also must consider the mechanisms by which estrogens and even androgens alter the activity of B cells to impact antibody responses, which are the primary correlate of protection from influenza.Consistent with observations of sex-specific effects of aging on antibody responses, we are now showing sex-specific effects of aging on numbers of antibody-producing cells, including GC B cells and plasmablasts, which should be considered during the design and dosing of seasonal and universal influenza vaccines.

Mice
Adult (8-10 weeks old) male and female C57BL/6CR mice were purchased from Charles River Laboratories (Frederick, MD), while the aged (17 months old) mice, originating from the Charles River Laboratories, were obtained from the National Institute on Aging.Dr. Arthur P. Arnold gifted breeder males for the FCG mouse model from the University of California, Los Angeles (46).The FCG mouse colony was maintained in-house by mating XY − males with wild-type C57BL/6 J females purchased from the Jackson Laboratory (Bar Harbor, ME).Genotypes were determined at weaning (i.e., at 3 weeks) by polymerase chain reaction (PCR) analysis for the presence or absence of the Sry gene as described (47).Pups of the same genotype were housed together and were used at 8-10 weeks of age.Mice were housed 5/cage under standard biosafety level-2 conditions in the Johns Hopkins Bloomberg School of Public Health animal facility with ad libitum food and water.All animal procedures were approved by the Johns Hopkins University Animal Care and Use Committee (MO20H236).

Antibody measurements
The levels of anti-2009 H1N1 IgG, IgG1, and IgG2c antibodies in plasma samples collected at different time points after vaccination were measured using our in-house enzyme-linked immunosorbent assays (ELISAs) (3,4,6).Briefly, plates were coated with 50 µL/well of sodium carbonate and sodium bicarbonate coating buffer contain ing 2 µg/mL of mouse-adapted 2009 H1N1 whole virus protein and were incubated overnight at 4°C.The next day, plates were washed three times, blocked with 10% skim milk solution for 1 h at 37°C, and then serially diluted plasma samples were added.After 1 h of incubation at 37°C, plates were washed, and horse-radish peroxidase-conju gated secondary IgG (Invitrogen), IgG1 (Invitrogen), and IgG2c antibodies (Invitrogen) were added.After 1 h of incubation at 37°C, plates were washed, and reactions were developed using 3,3′,5,5′-tetramethylbenzidine (BD Biosciences) for 20 min, stopped using 1 N hydrochloric acid (HCL).Plates were read at 450 nm wavelength using the ELISA plate reader (Molecular Devices), and the endpoint titer was calculated as the highest serum dilution with an average optical density (OD) value greater than three times the average OD of negative controls.Likewise, the virus-neutralizing antibody (nAb) titers on plasma samples, against the vaccine virus (i.e., ma2009 H1N1 virus), were measured using a Madin-Darby canine kidney (MDCK) cells-based microneutralization assay, as previously described (3,4,6).

Sex steroid measurement
Concentrations of sex steroids on plasma samples were measured using commercial testosterone (IBL America, Minneapolis, MN) and estradiol (Calbiotech Inc., El Cajon, CA) ELISA kits, as per the manufacturer's instructions (3,10).

Virus titration in lungs
For virus titration, lung samples collected at 3 or 5 dpc were homogenized, lung homogenates were 10-fold serially diluted in serum-free media and then transferred in six replicates in 96-well cell culture plates confluent with MDCK cells.Plates were incubated for 6 days at 32°C followed by fixation with 4% formaldehyde, staining with naphthol blue-black solution, and virus titer calculation by Reed and Muench method (6,10).

Somatic hypermutation
For SHM, splenic GC B cells (B220 + CD38 − GL7 + ) were sorted at 35 dpv using BD FACS Aria Fusion (BD Biosciences).Sorted cells were then lysed in a digestion buffer, genomic DNA was isolated by phenol/chloroform extraction and ethanol precipitation, and the J H 4 intronic region was amplified using a nested PCR protocol.The J H 4 intronic DNA (492 bp) was sequenced, and mutations in the unique VDJ clones were analyzed as described earlier (6).All sequences are publicly available at Mendeley Data (DOI: 10.17632/8f2xx4rxh9.1).

Statistical analysis
Data were analyzed in GraphPad Prism version 10.1.0.Sex steroids concentration, antibody titers, virus titers in the lungs, numbers of GC B cells and plasmablasts, and SHM frequencies were compared using two-way ANOVA followed by Tukey's multiple comparisons.Antibody responses up to 4 mpv and change in body mass after the virus challenge were compared using repeated measures ANOVA (mixed effects model) with Tukey's multiple comparisons.Data were considered statistically significant at P < 0.05.

FIG 1
FIG 1 The frequency and number of germinal center B cells and plasmablasts are greater in the draining lymph nodes and spleens from vaccinated adults but not aged, females than males.Adult (2-3 months old) and aged (17 months old) male and female C57BL/6CR mice were vaccinated twice with inactivated 2009 H1N1 vaccine in a 3-week interval.At 35 days post-vaccination (i.e., 14 days post-boost), draining lymph nodes and spleens were collected, single-cell suspensions were prepared, and flow cytometry was performed to measure the frequencies and numbers of GC B cells and plasmablasts.(A) Representative flow plots are shown from the lymph nodes of one adult female mouse.Total numbers of lymph node cells and total B cells were quantified (B and C).Frequencies and numbers of (D and E) GC B cells and (F and G) plasmablasts in the lymph nodes were quantified.The frequencies and numbers of GC B cells in the spleen were quantified (H, I), GC B cells were sorted, and (J) mutation frequency in the J H 4 intronic region of sorted splenic GC B cells was measured.Data represent the mean ± standard error of the mean (n = 5-19/group), and asterisks (*) represent significant differences (P < 0.05) between the groups based on two-way ANOVAs followed by Tukey's multiple comparisons tests in GraphPad Prism 10.1.0.

FIG 2
FIG 2 Adult, but not aged, females have higher antibody titers at 1-month post-vaccination.Adult (2-3 months old) and aged (17 months old) male and female C57BL/6CR mice were vaccinated twice with inactivated 2009 H1N1 vaccine in a 3-week interval.At 35 days post-vaccination (i.e., 1 mpv), plasma samples were collected to determine the titers of anti-2009 H1N1 influenza virus-specific (A) IgG, (B) IgG1, (C) IgG2c, and (D) virus-neutralizing antibody (nAb) titers and to measure the concentrations of (E) estradiol and (F) testosterone.Data represent the mean ± standard error of the mean (n = 5-20/group), and asterisks (*) represent significant differences (P < 0.05) between the groups based on two-way ANOVAs followed by Tukey's multiple comparisons tests in GraphPad Prism 10.1.0.

FIG 3
FIG 3 Adult female mice maintain higher titers of influenza vaccine-induced antibodies up to 4 months post-vaccination, which is mitigated with aging.Adult (2-3 months old) and aged (17 months old) male and female C57BL/6CR mice were vaccinated twice with inactivated 2009 H1N1 vaccine at a 3-week interval.Plasma samples were collected each month until 4 mpv, and anti-2009 H1N1 influenza virus-specific (A) IgG, (B) IgG2c, and (C) virus-neutralizing antibody (nAb) titers were measured.Data represent the mean ± standard error of the mean (n = 15-20/group), and significant differences between the groups are denoted by asterisks (*P < 0.05) based on repeated measures two-way ANOVAs followed by Tukey's multiple comparisons tests in GraphPad Prism 10.1.0.

FIG 4
FIG 4 Female-biased vaccine-induced protection against infection, but not disease, is maintained for up to 4 months post-vaccination (mpv) among adult, but not aged, animals.Adult (2-3 months old) and aged (17 months old) male and female C57BL/6CR mice were vaccinated twice with inactivated 2009 H1N1 vaccine at a 3-week interval.At 1 or 4 mpv, vaccinated mice were challenged with 10 5 TCID 50 of a drift variant of the 2009 H1N1 virus.(A and B) Replicating virus titers in the lungs were measured in a subset of mice at 5 days post-challenge (dpc), and (C and D) changes in body mass over a period of 14 dpc were measured in another subset of mice to compare protection from severe disease.Data represent the mean ± standard error of the mean (n = 5-10/group), and significant differences between the groups are denoted by asterisks (*P < 0.05) based on two-way ANOVAs or repeated measures two-way ANOVAs followed by Tukey's multiple comparisons tests in GraphPad Prism 10.1.0.

FIG 5
FIG 5 Gonadal sex more than sex chromosomal complement mediates influenza vaccine-induced immunity and protection.Eight-to 10-week-old four core genotype C57BL/6 J mice were vaccinated twice with the inactivated 2009 H1N1 vaccine at a 3-week interval.Plasma samples were collected at 28 days post-vaccination, and concentrations of (A) estradiol and (B) testosterone along with 2009 H1N1 influenza virus-specific (C) IgG, (D) IgG2c, and (E) virus-neutraliz ing antibody (nAb) titers were measured.At 35 dpv (i.e., 14 days post-boost), popliteal and inguinal lymph nodes were collected, single-cell suspensions were prepared, and the numbers of (F) germinal center B cells and (G) plasmablasts were quantified using flow cytometry.At 42 dpv, mice were challenged with 10 5 TCID 50 of a drift variant of the 2009 H1N1 virus, and (H) replicating virus titers in the lungs were measured in a subset of mice at 5 days post-challenge, and (I) the percentage change in body mass over a period of 14 dpc was measured in another subset of mice to evaluate protection from severe disease.Data represent the mean ± standard error of the mean (n = 5-27/group), and significant differences between the groups are denoted by asterisks (*P < 0.05) based on two-way ANOVAs or repeated measures two-way ANOVAs followed by Tukey's multiple comparisons tests in GraphPad Prism 10.1.0.

FIG 6
FIG 6 Estradiol replacement improves influenza vaccine-induced antibody responses and protection in aged female mice.Adult (2-3 months old) or aged (17 months old) female C57BL/6CR mice were subcutaneously implanted either with placebo or estradiol (E2)-loaded capsules.One week after capsule implantation, mice were vaccinated with inactivated 2009 H1N1 vaccine and boosted after 3 weeks.At 35 days post-vaccination, plasma samples were collected, and anti-2009 H1N1 influenza virus-specific (A) IgG, (B) IgG2c, and (C) virus-neutralizing antibody (nAb) titers were measured.At 42 dpv, vaccinated mice were challenged with 10 5 TCID 50 of a drift variant of the 2009 H1N1 virus.(D) Replicating virus titers in the lungs were measured in a subset of mice at 3 days post-challenge, and (E) changes in body mass over a period of 14 dpc were measured in another subset of mice to compare protection from severe disease.Data represent the mean ± standard error of the mean (n = 7-15/group), and significant differences between the groups are denoted by asterisks (*P < 0.05) based on two-way ANOVAs or repeated measures two-way ANOVAs followed by Tukey's multiple comparisons tests in GraphPad Prism 10.1.0.